Agarose is a natural polysaccharide extracted from algae, and its aqueous solution forms a hydrogel at low temperatures. Agarose has been used as chromatography medium since 1960s, and has many advantageous characteristics, such as high hydrophilicity, high porosity, and hydroxyl groups available for functionalization. Agarose is frequently used as a base matrix e.g. in affinity chromatography, hydrophobic interaction chromatography (HIC), reverse phase chromatography (RPC) and ion exchange chromatography.
For example, Shahab Lahooti, et al. (Shahab Lahooti and Michael V. Sefton, Effect of an immobilization matrix and capsule permeability on the variability of encapsulated HEK cells, Biomaterials. 21 (2000) 987-995) described agarose as core substance, surrounded by a hydroxyethyl methacrylate-methyl methacrylate copolymer shell to embed the HEK cells. On the one hand, the big pore net structure of agarose can evenly disperse cell and facilitate the diffusion of nutrient substances and metabolic products, on the other hand, it also reduces bead membrane concentration, increases membrane permeability, and facilitates sufficient nutrient substance entry to beads for cell growth. The research shows that agarose facilitates maintenance of the embedded cell activity and cell division and proliferation. The experiment results show that the cells proliferated twice as much in presence of agarose as in the absence of agarose after 14 days mainly because agarose disperses the cells evenly and offers supporting substrate to cells.
Hiroyuki Hayashi, et al. (Hiroyuki Hayashi, Kazutomo Inoue, Tun Aung et al, Application of a novel B cell line MIN6 to a mesh-reinforced polyvinyl alcohol hydrogel tube and three layer agarose microcapsules: An in vitro study, Cell Transplantation 5 (1996) S65-S69) described the use of agarose in the preparation of three-layer gel capsules used to embed B cell line MIN6. The research results show the embedded B cell line MIN6 has twice as high insulin secretion rate as unembedded MIN6.
Stellan Hjertén (Stellan Hjertén, The preparation of agarose spheres for chromatography of molecules and particles, Biochimia Et Biophysica Acta. 79 (1964) 393-398) described emulsification of agarose in an inverse suspension gelation method, using agitator emulsification.
Spraying methods using nozzles have been suggested (A. M. Egorev, A. Kh. Vakhabov and V. Ya. Chernyak, Isolation of agarose and granulation of agar and agarose gel, Journal of Chromatography. 46 (1970) 143-148; and S. Bengtsson and L. Philipsson, Chromatography of animal viruses on pearl-condensed agar, Biochimia Et Biophysica Acta. 79 (1964) 399-406) for the preparation of agarose beads as a separating medium or living cell carrier.
However, known drawbacks of such emulsion methods are that the particle size of the liquid droplets can not be controlled, the prepared emulsion has uneven particle size, the cured agarose gel beads have uneven particle size. In the separation process, small gel beads will flock to the gaps between gel beads to increase column back pressure and evenly cause no separation due to uneven particle size. When gel beads are used to the embed cells, each bead embeds a different number of cells and different proliferation rates occur during cell growth due to their uneven particle size. In addition, agarose gel beads with uniform particle size are very important to research gel properties. Uneven particle size will lead to complex characterization of beads. In addition, it is very difficult to control the particle size of the prepared beads and to prepare beads of small sizes such as below about 10 μm in these traditional preparation processes.
CN200410000087.9 describes traditional microporous membrane emulsification to prepare agarose gel beads with controllable uniform particle size (hereinafter denoted preparation of agarose gel beads using traditional membrane emulsification method). The traditional membrane emulsification can result in particles of even size. In this process, membranes having different pore diameters are selected to prepare beads with particle sizes in the range of 3-60 μm. Agarose gel beads with smaller particle sizes such as less than 10 μm are prepared with membranes having correspondingly small pore diameters. The emulsification rate is very slow at high nitrogen gas pressure. An increased pressure can increase the emulsification rate to a certain extent, but too high pressure was shown to reduce the particle size uniformity of beads. When beads have high agarose content, high pressure leads to severe broadening of the bead size distribution. This is considered a substantial drawback of this traditional method for certain applications, where both high agarose content and a small particle size are important.
During chromatographic separation and purification of biological molecules, it is an advantage if the separating medium can withstand high flow rates. Thus, some problems are known with the agarose medium widely used in biological separation field. As is well known, agarose gel structure is formed by mutual action of hydrogen bonds. At gelling state, polysaccharide chains will form a porous net structure through staggered hydrogen bonds between chains. This gel formed by non-covalent structure has low mechanical strength, and is consequently not capable of withstanding very high flow rates. The strength of agarose gel beads is increased using two methods.
U.S. Pat. No. 4,665,164 (Per-Åke Pernemalm, Mats Lindgren and Göran Lindgren. 1984. Polysaccharide crosslinked separation material and its preparation) relates to chemical crosslinking, namely that covalent bonds are introduced between hydroxyl groups on the polysaccharide chain to increase the mechanical strength of the gel. With a constant crosslink density, an alternative method is to increase the agarose content of the gel beads, by increasing the agarose solution concentration in the water phase. With increasing concentration of the agarose aqueous solution, however, its viscosity is increasing. In case of use of traditional membrane emulsification method to prepare agarose gel bead, the increasing viscosity of water phase brings about difficulties in emulsion preparation process. It is very difficult for the formed liquid droplets to detach from the membrane surface due to the higher viscosity of the water phase. In the case where small size liquid droplets are formed, these droplets will plug the membrane pores after a long emulsification process. The experiment results show that when traditional microporous membrane emulsification method is used to prepare gel beads with small particle size and high agarose content, high viscosity of the water phase leads to slow W/O emulsion preparation process even at high pressure.